Thank you for visiting nature.com. You are using a browser version with
limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off
compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site
without styles and JavaScript.

Subjects

Abstract

Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE–PACE) to improve their editing efficiency and target sequence compatibility. We used BE–PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.

Data availability

Key plasmids from this work will be available from Addgene (depositor, D.R.L.) and other plasmids are available upon request. All unmodified reads for sequencing-based data in the manuscript are available from the NCBI Sequence Read Archive, accession number PRJNA511456. Figures 4b, 5 and 6, Supplementary Table 3 and Supplementary Figs. 8–14, 16 and 18–22 are based on processing of sequencing data. Protein sequences used for Supplementary Fig. 17 are supplied as Supplementary Data 1.

Change history

12 August 2019

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Acknowledgements

We thank B. Fu and C. Canavan for assistance with plasmid construction and assays; H. Rees, T. Wang, J. Bessen, A. Badran and P. Lichtor for helpful discussion; K. Clement for CRISPResso2 support; and A. Hamidi for help editing the manuscript. This work was supported by US NIH grant nos. U01 AI142756, RM1 HG009490, R01 EB022376 and R35 GM118062, St. Jude Collaborative Research Consortium, DARPA HR0011-17-2-0049, the Ono Pharma Foundation and HHMI. L.W.K. is an NSF Graduate Research Fellow and was supported by NIH Training Grant no. T32 GM095450. O.S.O. and J.R.H. were supported by NIH DC013521. C.Z. was supported by the Harvard College Research Program. C.W. is the Marion Abbe Fellow of the Damon Runyon Cancer Research Foundation (DRG-2343-18).

Search for Mantu Bhaumik in:

Search for Olga Shubina-Oleinik in:

Search for Jeffrey R. Holt in:

Search for David R. Liu in:

Contributions

B.W.T. designed the research, designed and constructed plasmids, and performed PACE and bacterial experiments. L.W.K. and J.M.L. designed and performed HEK cell experiments and analyzed data. J.M.L. designed and performed APOE editing experiments. W-H.Y. designed and performed baringo editing experiments. C.Z. constructed plasmids and performed bacterial experiments for selection development. G.A.N. designed and constructed plasmids for the HEK cell experiment for WFS1 editing. C.W. designed and performed ancestral sequence reconstruction. M.B., O.S-O. and J.R.H. contributed baringo mouse cells. D.R.L. designed and supervised the research. B.W.T., L.W.K. and D.R.L. wrote the manuscript. All authors contributed to editing the manuscript.

Corresponding author

Ethics declarations

Competing interests

D.R.L. is a consultant and co-founder of Beam Therapeutics, Editas Medicine and Pairwise Plants, companies that use genome editing. D.R.L., B.W.T. and C.W. have filed patent applications on aspects of this work.